Subcloning analysis and generation of restriction maps are useful DNA analytical tools. In order to obtain them, one or more DNA fragments is/are partially digested to produce an array of restriction fragments. Partial digestion has also been used for the detection of polymorphisms of various known genetic loci in different organisms (1). Traditional partial digestions require either the termination of restriction digestion reactions at various time points or the use of a limiting restriction enzyme, (2) or the use of specific cloning vectors (3). A more sophisticated method is the combined use of dam methylase and Mbol to create partial digestion (4). A disadvantage is that the methylase method requires careful and detailed studies to determine the ratio of the two enzymes to be used for each combination of a DNA methylase and a restriction endonuclease.
The combination of PCR with 5-methyl-dCTP has been used to obtain substrates for assaying the sensitivity of different restriction enzymes towards fully methylated DNA templates (5). In this procedure, dCTP is fully replaced by 5-methyl dCTP in the polymerase chain reaction (PCR). which is not administered until after the PCR is complete. This method generates fully methylated PCR products. However, this method cannot produce a partially modified PCR product nor can it produce an array of DNA restriction fragments as in a partial digestion.
Accordingly, there is a need in the art of DNA analysis for a method requiring less enzyme specificity, and less time and material for making a partially modified PCR product and for producing the array of DNA restriction fragments.